ABSTRACT We use afferent sensory information to interact with the environment via senses like touch, vision, hearing, and taste. Sensory neurons rarely traverse epithelium, since epithelia form borders between our body and the environment. Thus, the central aspect of these senses is the neuro-epithelial connection, where a specialized epithelial cell (e.g. Merkel cell for touch, or hair cell for hearing) interacts with an afferent sensory neuron. Sensory neuro-epithelial connections are challenging to study because the soma of sensory neurons tend to be far away from the epithelia (e.g. DRG) and only specialized, sparsely distributed epithelial cells are capable of primary sensory transduction. Thus, despite its physiological importance, there remains little mechanistic understanding of neuro-epithelial communication. For example, every class of specialized sensory epithelial cells release a range of neuroactive transmitters, and primary afferent neurons express a variety of receptors and primary transduction proteins, providing a vast potential for redundant signaling pathways. Likewise, it is unclear whether neuro-epithelial communication occurs via synaptic, paracrine, or even exosome communication. Microfluidic devices in general provide an especially attractive reductionist system to study cell-to-cell communication. A microfluidics device that targets the study of neuro-epithelial communication has great potential to address the critical knowledge gap in understanding these important systems. The Co-PI of this proposal (Beyder) discovered a population of specialized mechanosensitive sensory epithelial cells in the gut that bears a close resemblance in development and function to the skin?s light touch sensors, the Merkel cells. These cells may signal to sensory neurons via synapse, paracrine, or endocrine routes. The gut neuro-epithelial connection has the advantage of being plentiful (the largest surface area between internal and external environments, and novel organoid techniques to generate epithelial organoids) and accessible (humans and animals can survive excision of both the epithelial cells and cell bodies of afferent neurons that are located in the gut wall). The long-term goal of the collaboration between the two Co-PIs (physician-scientist [Beyder] and biomedical engineer [Revzin]) is to develop novel tools to help delineate how biochemical and mechanical signals are integrated into the neuro-epithelial sensory system. Our hypothesis in this R21 project is that we can develop a microfluidic co-culture of enteric sensory neurons and specialized epithelial sensory cells that will recapitulate aspects of neuro-epithelial interactions observed in vivo. We will combine the ability to selectively stimulate the epithelial compartment by chemical or mechanical stimuli with the ability to guide neuronal extensions toward epithelial cells, and integrate genetically encoded optogenetic and chemogenetic sensory epithelial cell modulation and genetically-encoded calcium (Ca2+) reporters into neurons and epithelial cells to monitor the dynamics of signal propagation in response to stimuli.